Thermal tolerant avicelase from Acidothermus cellulolyticus

ABSTRACT

The invention provides a thermal tolerant (thermostable) cellulase, AviIII, that is a member of the glycoside hydrolase (GH) family. AviIII was isolated and characterized from  Acidothermus cellulolyticus  and, like many cellulases, the disclosed polypeptide and/or its derivatives may be useful for the conversion of biomass into biofuels and chemicals.

This application is a divisional application of another U.S. patentapplication Ser. No. 09/917,376 filed Jul. 28, 2001.

GOVERNMENT INTERESTS

The United States Government has rights in this invention under ContractNo. DE-AC36-99GO10337 between the United States Department of Energy andthe National Renewable Energy Laboratory, a Division of the MidwestResearch Institute.

FIELD OF THE INVENTION

The invention generally relates to a novel avicelase from Acidothermuscellulolyticus, AviIII. More specifically, the invention relates topurified and isolated AviIII polypeptides, nucleic acid moleculesencoding the polypeptides, and processes for production and use ofAviIII, as well as variants and derivatives thereof.

BACKGROUND OF THE INVENTION

Plant biomass as a source of energy production can include agriculturaland forestry products, associated by-products and waste, municipal solidwaste, and industrial waste. In addition, over 50 million acres in theUnited States are currently available for biomass production, and thereare a number of terrestrial and aquatic crops grown solely as a sourcefor biomass (A Wiselogel, et al. Biomass feedstocks resources andcomposition—in C E Wyman, ed. Handbook on Bioethanol: Production andUtilization. Washington, D.C.: Taylor & Francis, 1996, pp 105-118).Biofuels produced from biomass include ethanol, methanol, biodiesel, andadditives for reformulated gasoline. Biofuels are desirable because theyadd little, if any, net carbon dioxide to the atmosphere and becausethey greatly reduce ozone formation and carbon monoxide emissions ascompared to the environmental output of conventional fuels. (P Bergeron.Environmental impacts of bioethanol—in C E Wyman, ed. Handbook onBioethanol: Production and Utilization. Washington, D.C.: Taylor &Francis, 1996, pp 90-103).

Plant biomass is the most abundant source of carbohydrate in the worlddue to the lignocellulosic materials composing the cell walls of allhigher plants. Plant cell walls are divided into two sections, theprimary and the secondary cell walls. The primary cell wall, whichprovides structure for expanding cells (and hence changes as the cellgrows), is composed of three major polysaccharides and one group ofglycoproteins. The predominant polysaccharide, and most abundant sourceof carbohydrates, is cellulose, while hemicellulose and pectin are alsofound in abundance. Cellulose is a linear beta-(1,4)-D-glucan andcomprises 20% to 30% of the primary cell wall by weight. The secondarycell wall, which is produced after the cell has completed growing, alsocontains polysaccharides and is strengthened through polymeric lignincovalently cross-linked to hemicellulose.

Carbohydrates, and cellulose in particular can be converted to sugars bywell-known methods including acid and enzymatic hydrolysis. Enzymatichydrolysis of cellulose requires the processing of biomass to reducesize and facilitate subsequent handling. Mild acid treatment is thenused to hydrolyze part or all of the hemicellulose content of thefeedstock. Finally, cellulose is converted to ethanol through theconcerted action of cellulases and saccharolytic fermentation(simultaneous saccharification fermentation (SSF)). The SSF process,using the yeast Saccharomyces cerevisiae for example, is oftenincomplete, as it does not utilize the entire sugar content of the plantbiomass, namely the hemicellulose fraction.

The cost of producing ethanol from biomass can be divided into threeareas of expenditure: pretreatment costs, fermentation costs, and othercosts. Pretreatment costs include biomass milling, pretreatmentreagents, equipment maintenance, power and water, and wasteneutralization and disposal. The fermentation costs can include enzymes,nutrient supplements, yeast, maintenance and scale-up, and wastedisposal. Other costs include biomass purchase, transportation andstorage, plant labor, plant utilities, ethanol distillation, andadministration (which may include technology-use licenses). One of themajor expenses incurred in SSF is the cost of the enzymes, as about onekilogram of cellulase is required to fully digest 50 kilograms ofcellulose. Economical production of cellulase is also compounded byfactors such as the relatively slow growth rates of cellulase-producingorganisms, levels of cellulase expression, and the tendency ofenzyme-dependent processes to partially or completely inactivate enzymesdue to conditions such as elevated temperature, acidity, proteolyticdegradation, and solvent degradation.

Enzymatic degradation of cellulose requires the coordinate action of atleast three different types of cellulases. Such enzymes are given anEnzyme Commission (EC) designation according to the NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology (Eur. J. Biochem. 264: 607-609 and 610-650, 1999).Endo-beta-(1,4)-glucanases (EC 3.2.1.4) cleave the cellulose strandrandomly along its length, thus generating new chain ends.Exo-beta-(1,4)-glucanases (EC 3.2.1.91) are processive enzymes andcleave cellobiosyl units (beta-(1,4)-glucose dimers) from free ends ofcellulose strands. Lastly, beta-D-glucosidases (cellobiases: EC3.2.1.21) hydrolyze cellobiose to glucose. All three of these generalactivities are required for efficient and complete hydrolysis of apolymer such as cellulose to a subunit, such as the simple sugar,glucose.

Highly thermostable enzymes have been isolated from the cellulolyticthermophile Acidothermus cellulolyticus gen. nov., sp. nov., a bacteriumoriginally isolated from decaying wood in an acidic, thermal pool atYellowstone National Park. A. Mohagheghi et al., (1986) Int. J.Systematic Bacteriology, 36(3): 435-443. One cellulase enzyme producedby this organism, the endoglucanase EI, is known to display maximalactivity at 75° C. to 83° C. M. P. Tucker et al. (1989), Bio/Technology,7(8): 817-820. E1 endoglucanase has been described in U.S. Pat. No.5,275,944. The A. cellulolyticus E1 endoglucanase is an activecellulase; in combination with the exocellulase CBH I from Trichodermareesei, E1 gives a high level of saccharification and contributes to adegree of synergism. Baker J O et al. (1994), Appl. Biochem.Biotechnol., 45/46: 245-256. The gene coding EI catalytic andcarbohydrate binding domains and linker peptide were described in U.S.Pat. No. 5,536,655. E1 has also been expressed as a stable, activeenzyme from a wide variety of hosts, including E. coli, Streptomyceslividans, Pichia pastoris, cotton, tobacco, and Arabidopsis (Dai Z,Hooker B S, Anderson D B, Thomas S R. Transgenic Res. 2000 February;9(1):43-54).

There is a need within the art to generate alternative cellulase enzymescapable of assisting in the commercial-scale processing of cellulose tosugar for use in biofuel production. Against this backdrop the presentinvention has been developed. The potential exists for the successful,commercial-scale expression of heterologous cellulase polypeptides, andin particular novel cellulase polypeptides with or without any one ormore desirable properties such as thermal tolerance, and partial orcomplete resistance to extreme pH inactivation, proteolyticinactivation, solvent inactivation, chaotropic agent inactivation,oxidizing agent inactivation, and detergent inactivation. Suchexpression can occur in fungi, bacteria, and other hosts.

SUMMARY OF THE INVENTION

The present invention provides AviIII, a novel member of the glycosidehydrolase (GH) family of enzymes, and in particular a thermal tolerantglycoside hydrolase useful in the degradation of cellulose. AviIIIpolypeptides of the invention include those having an amino acidsequence shown in SEQ ID NO:1, as well as polypeptides havingsubstantial amino acid sequence identity to the amino acid sequence ofSEQ ID NO:1 and useful fragments thereof, including, a catalytic domainhaving significant sequence similarity to the GH74 family, acarbohydrate binding domain (type III). See FIG. 1.

The invention also provides a polynucleotide molecule encoding AviIIIpolypeptides and fragments of AviIII polypeptides, for example catalyticand carbohydrate binding domains. Polynucleotide molecules of theinvention include those molecules having a nucleic acid sequence asshown in SEQ ID NO:2; those that hybridize to the nucleic acid sequenceof SEQ ID NO:2 under high stringency conditions; and those havingsubstantial nucleic acid identity with the nucleic acid sequence of SEQID NO:2.

The invention includes variants and derivatives of the AviIIIpolypeptides, including fusion proteins. For example, fusion proteins ofthe invention include AviIII polypeptide fused to a heterologous proteinor peptide that confers a desired function. The heterologous protein orpeptide can facilitate purification, oligomerization, stabilization, orsecretion of the AviIII polypeptide, for example. As further examples,the heterologous polypeptide can provide enhanced activity, includingcatalytic or binding activity, for AviIII polypeptides, where theenhancement is either additive or synergistic. A fusion protein of anembodiment of the invention can be produced, for example, from anexpression construct containing a polynucleotide molecule encodingAviIII polypeptide in frame with a polynucleotide molecule for theheterologous protein. Embodiments of the invention also comprisevectors, plasmids, expression systems, host cells, and the like,containing a AviIII polynucleotide molecule. Genetic engineering methodsfor the production of AviIII polypeptides of embodiments of theinvention include expression of a polynucleotide molecule in cell freeexpression systems and in cellular hosts, according to known methods.

The invention further includes compositions containing a substantiallypurified AviIII polypeptide of the invention and a carrier. Suchcompositions are administered to a biomass containing cellulose for thereduction or degradation of the cellulose.

The invention also provides reagents, compositions, and methods that areuseful for analysis of AviIII activity.

These and various other features as well as advantages whichcharacterize the present invention will be apparent from a reading ofthe following detailed description and a review of the associateddrawings.

The following Tables 1 and 2 includes sequences used in describingembodiments of the present invention. In Table 1, the abbreviations areas follows: CD, catalytic domain; CBD_III, carbohydrate binding domaintype III. When used herein, N* indicates a string of unknown nucleicacid units, and X* indicates a string of unknown amino acid units, forexample about 50 or more. Table 1 includes approximate start and stopinformation for segments, and Table 2 includes amino acid sequence datafor segments.

TABLE 1 Nucleotide and polypeptide segments. aa AviIII base base Length,BEGIN aa Length, SEQ ID No. SEQ ID No. Segment BEGIN END bp No. aa ENDNo. aa aa (amino acid) (nucleotide) Total 1 about about 3 1 M about X*about 1 1 2 length 3000 kb 1000 1000 Signal 31  138  108 11 M  46 A  36(potential) CD (GH74) 139 2358 2220 47 A  786 G 740 3 CBD_III 2607 aboutabout 0.5 869 V about X* about 4 (partial) 3000 kb 1000 154 CBD_III 26072838  264 869 V  956 Q  88 5 (partial)

TABLE 2 Gene/polypeptide segments with amino acid sequences. SEQ ID No.SEQ ID No. AviIII (amino acid) (nucleotide) Segment Segment Data 1 2Total length SEQ ID NO:1 (see Table 3); SEQ ID NO:2 (see Table 4) 8Signal (potential) MRSRRLVSLLAATASFAVAAALGVLPIAITASPAHA 3 CD (GH74)ATTQPYTWSNVAIGGGGFVDGIVFNEGAPGILYVRTDIGGMYRWDAANGRWIPLLDWVGWNNWGYNGVVSIAADPINTNKVWAAVGMYTNSWDPNDGAILRSSDQGATWQITPLPFKLGGNMPGRGMGERLAVDPNNDNILYFGAPSGKGLWRSTDSGATWSQMTNFPDVGTYIANPTDTTGYQSDIQGVVWVAFDKSSSSLGQASKTIFVGVADPNNPVFWSRDGGATWQAVPGAPTGFIPHKGVFDPVNHVLYIATSNTGGPYDGSSGDVWKFSVTSGTWTRISPVPSTDTANDYFGYSGLTIDRQHPNTIMVATQISWWPDTIIFRSTDGGATWTRIWDWTSYPNRSLRYVLDISAEPWLTFGVQPNPPVPSPKLGWMDEAMAIDPFNSDRMLYGTGATLYATNDLTKWDSGGQIHIAPMVKGLEETAVNDLISPPSGAPLISALGDLGGFTHADVTAVPSTIFTSPVFTTGTSVDYAELNPSIIVRAGSFDPSSQPNDRHVAFSTDGGKNWFQGSEPGGVTTGGTVAASADGSRFVWAPGDPGQPVVYAVGFGNSWAASQGVPANAQIRSDRVNPKTFYALSNGTFYRSTDGGVTFQPVAAGLPSSGAVGVMFHAVPGKEGDLWLAASSGLYHSTNGGSSWSAITGVSSAVNVGFGKSAPGSSYPAVFVVGTIGGVTGAYRSDDCGTTWVLINDDQHQYGNWGQAITGDHANLRRVYIGTNGRGIVYGDIGGAPSG 4 CBD_IIIVSGGVKVQYKNNDSAPGDNQIKPGLQVVNTGSSSVDLSTVTVRYWFTRDGGSSTLVYNCDWAA(partial) IGCGNIRASFGSVNPATPTADTYLQX* 5 CBD_IIIVSGGVKVQYKNNDSAPGDNQIKPGLQVVNTGSSSVDLSTVTVRYWFTRDGGSSTLVYNCDWAA(partial) IGCGNIRASFGSVNPATPTADTYLQ

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the gene sequence and amino acidsegment organization.

FIG. 2 is a graphic representation of the glycoside hydrolasegene/protein families found in various organisms.

DETAILED DESCRIPTION Definitions

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present disclosure:

“Amino acid” refers to any of the twenty naturally occurring amino acidsas well as any modified amino acid sequences. Modifications may includenatural processes such as posttranslational processing, or may includechemical modifications which are known in the art. Modifications includebut are not limited to: phosphorylation, ubiquitination, acetylation,amidation, glycosylation, covalent attachment of flavin,ADP-ribosylation, cross linking, iodination, methylation, and alike.

“Antibody” refers to a Y-shaped molecule having a pair of antigenbinding sites, a hinge region and a constant region. Fragments ofantibodies, for example an antigen binding fragment (Fab), chimericantibodies, antibodies having a human constant region coupled to amurine antigen binding region, and fragments thereof, as well as otherwell known recombinant antibodies are included in the present invention.

“Antisense” refers to polynucleotide sequences that are complementary totarget “sense” polynucleotide sequence.

“Binding activity” refers to any activity that can be assayed bycharacterizing the ability of a polypeptide to bind to a substrate. Thesubstrate can be a polymer such as cellulose or can be a complexmolecule or aggregate of molecules where the entire moiety comprises atleast some cellulose.

“Cellulase activity” refers to any activity that can be assayed bycharacterizing the enzymatic activity of a cellulase. For example,cellulase activity can be assayed by determining how much reducing sugaris produced during a fixed amount of time for a set amount of enzyme(see Irwin et al., (1998) J. Bacteriology, 1709-1714). Other assays arewell known in the art and can be substituted.

“Complementary” or “complementarity” refers to the ability of apolynucleotide in a polynucleotide molecule to form a base pair withanother polynucleotide in a second polynucleotide molecule. For example,the sequence A-G-T is complementary to the sequence T-C-A.Complementarity may be partial, in which only some of thepolynucleotides match according to base pairing, or complete, where allthe polynucleotides match according to base pairing.

“Expression” refers to transcription and translation occurring within ahost cell. The level of expression of a DNA molecule in a host cell maybe determined on the basis of either the amount of corresponding mRNAthat is present within the cell or the amount of DNA molecule encodedprotein produced by the host cell (Sambrook et al., 1989, Molecularcloning: A Laboratory Manual, 18.1-18.88).

“Fusion protein” refers to a first protein having attached a second,heterologous protein. Preferably, the heterologous protein is fused viarecombinant DNA techniques, such that the first and second proteins areexpressed in frame. The heterologous protein can confer a desiredcharacteristic to the fusion protein, for example, a detection signal,enhanced stability or stabilization of the protein, facilitatedoligomerization, of the protein, or facilitated purification of thefusion protein. Examples of heterologous proteins useful in the fusionproteins of the invention include molecules having one or more catalyticdomains of AviIII, one or more binding domains of AviIII, one or morecatalytic domains of a glycoside hydrolase other than AviIII, one ormore binding domains of a glycoside hydrolase other than AviIII, or anycombination thereof. Further examples include immunoglobulin moleculesand portions thereof, peptide tags such as histidine tag (6-His),leucine zipper, substrate targeting moieties, signal peptides, and thelike. Fusion proteins are also meant to encompass variants andderivatives of AviIII polypeptides that are generated by conventionalsite-directed mutagenesis and more modern techniques such as directedevolution, discussed infra.

“Genetically engineered” refers to any recombinant DNA or RNA methodused to create a prokaryotic or eukaryotic host cell that expresses aprotein at elevated levels, at lowered levels, or in a mutated form. Inother words, the host cell has been transfected, transformed, ortransduced with a recombinant polynucleotide molecule, and thereby beenaltered so as to cause the cell to alter expression of the desiredprotein. Methods and vectors for genetically engineering host cells arewell known in the art; for example various techniques are illustrated inCurrent Protocols in Molecular Biology, Ausubel et al., eds. (Wiley &Sons, New York, 1988, and quarterly updates). Genetically engineeringtechniques include but are not limited to expression vectors, targetedhomologous recombination and gene activation (see, for example, U.S.Pat. No. 5,272,071 to Chappel) and trans activation by engineeredtranscription factors (see, for example, Segal et al., 1999, Proc NatlAcad Sci USA 96(6):2758-63).

“Glycoside hydrolase family” refers to a family of enzymes whichhydrolyze the glycosidic bond between two or more carbohydrates orbetween a carbohydrate and a non-carbohydrate moiety (Henrissat B.,(1991) Biochem. J., 280:309-316). Identification of a putative glycosidehydrolase family member is made based on an amino acid sequencecomparison and the finding of significant sequence similarity within theputative member's catalytic domain, as compared to the catalytic domainsof known family members.

“Homology” refers to a degree of complementarity betweenpolynucleotides, having significant effect on the efficiency andstrength of hybridization between polynucleotide molecules. The termalso can refer to a degree of similarity between polypeptides.

“Host cell” or “host cells” refers to cells expressing a heterologouspolynucleotide molecule. Host cells of the present invention expresspolynucleotides encoding AviIII or a fragment thereof. Examples ofsuitable host cells useful in the present invention include, but are notlimited to, prokaryotic and eukaryotic cells. Specific examples of suchcells include bacteria of the genera Escherichia, Bacillus, andSalmonella, as well as members of the genera Pseudomonas, Streptomyces,and Staphylococcus; fungi, particularly filamentous fungi such asTrichoderma and Aspergillus, Phanerochaete chrysosporium and other whiterot fungi; also other fungi including Fusaria, molds, and yeastincluding Saccharomyces sp., Pichia sp., and Candida sp. and the like;plants e.g. Arabidopsis, cotton, barley, tobacco, potato, and aquaticplants and the like; SF9 insect cells (Summers and Smith, 1987, TexasAgriculture Experiment Station Bulletin, 1555), and the like. Otherspecific examples include mammalian cells such as human embyonic kidneycells (293 cells), Chinese hamster ovary (CHO) cells (Puck et al., 1958,Proc. Natl. Acad. Sci. USA 60, 1275-1281), human cervical carcinomacells (HELA) (ATCC CCL 2), human liver cells (Hep G2) (ATCC HB8065),human breast cancer cells (MCF-7) (ATCC HTB22), human colon carcinomacells (DLD-1) (ATCC CCL 221), Daudi cells (ATCC CRL-213), murine myelomacells such as P3/NSI/1-Ag4-1 (ATCC TIB-18), P3X63Ag8 (ATCC TIB-9),SP2/0-Ag14 (ATCC CRL-1581) and the like.

“Hybridization” refers to the pairing of complementary polynucleotidesduring an annealing period. The strength of hybridization between twopolynucleotide molecules is impacted by the homology between the twomolecules, stringency of the conditions involved, the meltingtemperature of the formed hybrid and the G:C ratio within thepolynucleotides.

“Identity” refers to a comparison between pairs of nucleic acid or aminoacid molecules. Methods for determining sequence identity are known.See, for example, computer programs commonly employed for this purpose,such as the Gap program (Wisconsin Sequence Analysis Package, Version 8for Unix, Genetics Computer Group, University Research Park, MadisonWis.), that uses the algorithm of Smith and Waterman, 1981, Adv. Appl.Math., 2: 482-489.

“Isolated” refers to a polynucleotide or polypeptide that has beenseparated from at least one contaminant (polynucleotide or polypeptide)with which it is normally associated. For example, an isolatedpolynucleotide or polypeptide is in a context or in a form that isdifferent from that in which it is found in nature.

“Nucleic acid sequence” refers to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alonga polypeptide chain. The deoxyribonucleotide sequence thus codes for theamino acid sequence.

“Polynucleotide” refers to a linear sequence of nucleotides. Thenucleotides may be ribonucleotides, or deoxyribonucleotides, or amixture of both. Examples of polynucleotides in the context of thepresent invention include single and double stranded DNA, single anddouble stranded RNA, and hybrid molecules having mixtures of single anddouble stranded DNA and RNA. The polynucleotides of the presentinvention may contain one or more modified nucleotides.

“Protein,” “peptide,” and “polypeptide” are used interchangeably todenote an amino acid polymer or a set of two or more interacting orbound amino acid polymers.

“Purify,” or “purified” refers to a target protein that is free from atleast 5-10% of contaminating proteins. Purification of a protein fromcontaminating proteins can be accomplished using known techniques,including ammonium sulfate or ethanol precipitation, acid precipitation,heat precipitation, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography, size-exclusionchromatography, and lectin chromatography. Various protein purificationtechniques are illustrated in Current Protocols in Molecular Biology,Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterlyupdates).

“Selectable marker” refers to a marker that identifies a cell as havingundergone a recombinant DNA or RNA event. Selectable markers include,for example, genes that encode antimetabolite resistance such as theDHFR protein that confers resistance to methotrexate (Wigler et al,1980, Proc Natl Acad Sci USA 77:3567; O'Hare et al., 1981, Proc NatlAcad Sci USA, 78:1527), the GPT protein that confers resistance tomycophenolic acid (Mulligan & Berg, 1981, PNAS USA, 78:2072), theneomycin resistance marker that confers resistance to the aminoglycosideG-418 (Calberre-Garapin et al., 1981, J Mol Biol, 150:1), the Hygroprotein that confers resistance to hygromycin (Santerre et al., 1984,Gene 30:147), and the Zeocin™ resistance marker (Invitrogen). Inaddition, the herpes simplex virus thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes can be employed in tk⁻, hgprt⁻ and aprt⁻cells, respectively.

“Stringency” refers to the conditions (temperature, ionic strength,solvents, etc) under which hybridization between polynucleotides occurs.A hybridzation reaction conducted under high stringency conditions isone that will only occur between polynucleotide molecules that have ahigh degree of complementary base pairing (85% to 100% identity).Conditions for high stringency hybridization, for example, may includean overnight incubation at about 42° C. for about 2.5 hours in6×SSC/0.1% SDS, followed by washing of the filters in 1.0×SSC at 65° C.,0.1% SDS. A hybridization reaction conducted under moderate stringencyconditions is one that will occur between polynucleotide molecules thathave an intermediate degree of complementary base pairing (50% to 84%identity).

“Substrate targeting moiety” refers to any signal on a substrate, eithernaturally occurring or genetically engineered, used to target any AviIIIpolypeptide or fragment thereof to a substrate. Such targeting moietiesinclude ligands that bind to a substrate structure. Examples ofligand/receptor pairs include carbohydrate binding domains andcellulose. Many such substrate-specific ligands are known and are usefulin the present invention to target an AviIII polypeptide or fragmentthereof to a substrate. A novel example is an AviIII carbohydratebinding domain that is used to tether other molecules to acellulose-containing substrate such as a fabric.

“Thermal tolerant” refers to the property of withstanding partial orcomplete inactivation by heat and can also be described as thermalresistance or thermal stability. Although some variation exists in theliterature, the following definitions can be considered typical for theoptimum temperature range of stability and activity for enzymes:psychrophilic (below freezing to 10 C); mesophilic (10° C. to 50° C.);thermophilic (50° C. to 75° C.); and caldophilic (75° C. to aboveboiling water temperature). The stability and catalytic activity ofenzymes are linked characteristics, and the ways of measuring theseproperties vary considerably. For industrial enzymes, stability andactivity are best measured under use conditions, often in the presenceof substrate. Therefore, cellulases that must act on process streams ofcellulose must be able to withstand exposure up to thermophilic or evencaldophilic temperatures for digestion times in excess of several hours.

In encompassing a wide variety of potential applications for embodimentsof the present invention, thermal tolerance refers to the ability tofunction in a temperature range of from about 15° C. to about 100° C. Apreferred range is from about 30° C. to about 80° C. A highly preferredrange is from about 50° C. to about 70° C. For example, a protein thatcan function at about 45° C. is considered in the preferred range eventhough it may be susceptible to partial or complete inactivation attemperatures in a range above about 45° C. and less than about 80° C.For polypeptides derived from organisms such as Acidothermus, thedesirable property of thermal tolerance among is often accompanied byother desirable characteristics such as: resistance to extreme pHdegradation, resistance to solvent degradation, resistance toproteolytic degradation, resistance to detergent degradation, resistanceto oxidizing agent degradation, resistance to chaotropic agentdegradation, and resistance to general degradation. Cowan D A in DansonM J et al. (1992) The Archaebacteria, Biochemistry and Biotechnology at149-159, University Press, Cambridge, ISBN 1855780100. Here ‘resistance’is intended to include any partial or complete level of residualactivity. When a polypeptide is described as thermal tolerant it isunderstood that any one, more than one, or none of these other desirableproperties can be present.

“Variant”, as used herein, means a polynucleotide or polypeptidemolecule that differs from a reference molecule. Variants can includenucleotide changes that result in amino acid substitutions, deletions,fusions, or truncations in the resulting variant polypeptide whencompared to the reference polypeptide.

“Vector,” “extra-chromosomal vector” or “expression vector” refers to afirst polynucleotide molecule, usually double-stranded, which may haveinserted into it a second polynucleotide molecule, for example a foreignor heterologous polynucleotide. The heterologous polynucleotide moleculemay or may not be naturally found in the host cell, and may be, forexample, one or more additional copy of the heterologous polynucleotidenaturally present in the host genome. The vector is adapted fortransporting the foreign polynucleotide molecule into a suitable hostcell. Once in the host cell, the vector may be capable of integratinginto the host cell chromosomes. The vector may optionally containadditional elements for selecting cells containing the integratedpolynucleotide molecule as well as elements to promote transcription ofmRNA from transfected DNA. Examples of vectors useful in the methods ofthe present invention include, but are not limited to, plasmids,bacteriophages, cosmids, retroviruses, and artificial chromosomes.

Within the application, unless otherwise stated, the techniques utilizedmay be found in any of several well-known references, such as: MolecularCloning: A Laboratory Manual (Sambrook et al. (1989) Molecular cloning:A Laboratory Manual), Gene Expression Technology (Methods in Enzymology,Vol. 185, edited by D. Goeddel, 1991 Academic Press, San Diego, Calif.),“Guide to Protein Purification” in Methods in Enzymology (M. P.Deutshcer, 3d., (1990) Academic Press, Inc.), PCR Protocols: A Guide toMethods and Applications (Innis et al. (1990) Academic Press, San Diego,Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2^(nd)ed. (R. I. Freshney (1987) Liss, Inc., New York, N.Y.), and GeneTransfer and Expression Protocols, pp 109-128, ed. E. J. Murray, TheHumana Press Inc., Clifton, N.J.).

O-Glycoside Hydrolases:

Glycoside hydrolases are a large and diverse family of enzymes thathydrolyse the glycosidic bond between two carbohydrate moieties orbetween a carbohydrate and a non-carbohydrate moiety (See FIG. 2).Glycoside hydrolase enzymes are classified into glycoside hydrolase (GH)families based on significant amino acid similarities within theircatalytic domains. Enzymes having related catalytic domains are groupedtogether within a family, (Henrissat et al., (1991) supra, and Henrissatet al. (1996), Biochem. J. 316:695-696), where the underlyingclassification provides a direct relationship between the GH domainamino acid sequence and how a GH domain will fold. This informationultimately provides a common mechanism for how the enzyme will hydrolysethe glycosidic bond within a substrate, i.e., either by a retainingmechanism or inverting mechanism (Henrissat., B, (1991) supra).

Cellulases belong to the GH family of enzymes. Cellulases are producedby a variety of bacteria and fungi to degrade the beta-(1,4)-glycosidicbond of cellulose and to so produce successively smaller fragments ofcellulose and ultimately produce glucose. At present, cellulases arefound within are at least 11 different GH families. Three differenttypes of cellulase enzyme activities have been identified within theseGH families: exo-acting cellulases which cleave successive disaccharideunits from the non-reducing ends of a cellulose chain; endo-actingcellulases which randomly cleave successive disaccharide units withinthe cellulose chain; and β-glucosidases which cleave successivedisaccharide units to glucose (J. W. Deacon, (1997) Modern Mycology, 3rdEd., ISBN: 0-632-03077-1, 97-98).

Many cellulases are characterized by having a multiple domain unitwithin their overall structure, a GH or catalytic domain is joined to acarbohydrate-binding domain (CBD) by a glycosylated linker peptide(Koivula et al., (1996) Protein Expression and Purification 8:391-400).As noted above, cellulases do not belong to any one family of GHdomains, but rather have been identified within at least 11 different GHfamilies to date. The CBD type domain increases the concentration of theenzyme on the substrate, in this case cellulose, and the linker peptideprovides flexibility for both larger domains.

Conversion of cellulose to glucose is an essential step in theproduction of ethanol or other biofuels from biomass. Cellulases are animportant component of this process, where approximately one kilogram ofcellulase can digest fifty kilograms of cellulose. Within this process,thermostable cellulases have taken precedent, due to their ability tofunction at elevated temperatures and under other conditions includingpH extremes, solvent presence, detergent presence, proteolysis, etc.(see Cowan D A (1992), supra).

Highly thermostable cellulase enzymes are secreted by the cellulolyticthemophile Acidothermus cellulolyticus (U.S. Pat. Nos. 5,275,944 and5,110,735). This bacterium was originally isolated from decaying wood inan acidic, thermal pool at Yellowstone National Park and deposited withthe American Type Culture Collection (ATCC 43068) (Mohagheghi et al.,(1986) Int. J. System. Bacteriol., 36:435-443).

Recently, a thermostable cellulase, E1 endoglucanase, was identified andcharacterized from Acidothermus cellulolyticus (U.S. Pat. No.5,536,655). The E1 endoglucanase has maximal activity between 75 and 83°C. and is active to a pH well below 5. Thermostable cellulase, and E1endoglucanase, are useful in the conversion of biomass to biofuels, andin particular, are useful in the conversion of cellulose to glucose.Conversion of biomass to biofuel represents an extremely importantalternative fuel source that is more environmentally friendly thanconventional fuels, and provides a use, in some cases, for wasteproducts.

AviIII:

As described more fully in the Examples below, AviIII, a novelthermostable cellulase, has now been identified and characterized. Thepredicted amino acid sequence of AviIII (SEQ ID NO:1) has anorganization characteristic of a cellulase enzyme. AviIII contains acatalytic domain—carbohydrate binding domain unit. In particular, AviIIIincludes a GH74 catalytic domain (amino acids from about A47 to aboutG786), and a carbohydrate binding domain type III (CBDIII) (amino acidsfrom about V869 to about at least Q956).

As discussed in more detail below (Example 2), significant amino acidsimilarity of AviIII to other cellulases identifies AviIII as acellulase. In addition, the predicted amino acid sequence (SEQ ID NO: 1)indicates that a CBD type III domain is present as characterized byTomme P. et al. (1995), in Enzymatic Degradation of InsolublePolysaccharides (Saddler J N & Penner M, eds.), at 142-163, AmericanChemical Society, Washington. See also Tomme, P. & Claeyssens, M. (1989)FEBS Lett. 243, 239-243; Gilkes, N. R et al., (1988) J. Biol. Chem. 263,10401-10407.

AviIII, as noted above, has a catalytic domain, identified as belongingto the GH74 family. The GH74 domain family includes an avicelase fromAspergillus aculeatus.

AvIII is also a thermostable cellulase as it is produced by thethemophile Acidothermus cellulolyticus. As discussed, AviIIIpolypeptides can have other desirable characteristics (see Cowan D A(1992), supra). Like other members of the cellulase family, and inparticular thermostable cellulases, AviIII polypeptides are useful inthe conversion of biomass to biofuels and biofuel additives, and inparticular, biofuels from cellulose. It is envisioned that AviIIIpolypeptides could be used for other purposes, for example indetergents, pulp and paper processing, food and feed processing, and intextile processes. AviIII polypeptides can be used alone or incombination with one or more other cellulases or glycoside hydrolases toperform the uses described herein or known within the relevant art, allof which are within the scope of the present disclosure.

AviIII Polypeptides:

AviIII polypeptides of the invention include isolated polypeptideshaving an amino acid sequence as shown below in Example 1; Table 3 andin SEQ ID NO:1, as well as variants and derivatives, includingfragments, having substantial identity to the amino acid sequence of SEQID NO:1 and that retain any of the functional activities of AviIII.AviIII polypeptide activity can be determined, for example, bysubjecting the variant, derivative, or fragment to a substrate bindingassay or a cellulase activity assay such as those described in Irwin Det al., J. Bacteriology 180(7): 1709-1714 (April 1998).

TABLE 3 AviIII amino acid sequence. (SEQ ID NO:1)MDRSENIRLTMRSRRLVSLLAATASFAVAAALGVLPIAITASPAHAATTQPYTWSNVAIGGGGFVDGIVFNEGAPGILYVRTDIGGMYRWDAANGRWIPLLDWVGWNNWGYNGVVSIAADPINTNKVWAAVGMYTNSWDPNDGAILRSSDQGATWQITPLPFKLGGNMPGRGMGERLAVDPNNDNILYFGAPSGKGLWRSTDSGATWSQMTNFPDVGTYIANPTDTTGYQSDIQGVVWVAFDKSSSSLGQASKTIFVGVADPNNPVFWSRDGGATWQAVPGAPTGFIPHKGVFDPVNHVLYIATSNTGGPYDGSSGDVWKFSVTSGTWTRISPVPSTDTANDYFGYSGLTIDRQHPNTIMVATQISWWPDTIIFRSTDGGATWTRIWDWTSYPNRSLRYVLDISAEPWLTFGVQPNPPVPSPKLGWMDEAMAIDPFNSDRMLYGTGATLYATNDLTKWDSGGQIHIAPMVKGLEETAVNDLISPPSGAPLISALGDLGGFTHADVTAVPSTIFTSPVFTTGTSVDYAELNPSIIVRAGSFDPSSQPNDRHVAFSTDGGKNWFQGSEPGGVTTGGTVAASADGSRFVWAPGDPGQPVVYAVGFGNSWAASQGVPANAQIRSDRVNPKTFYALSNGTFYRSTDGGVTFQPVAAGLPSSGAVGVMFHAVPGKEGDLWLAASSGLYHSTNGGSSWSAITGVSSAVNVGFGKSAPGSSYPAVFVVGTIGGVTGAYRSDDCGTTWVLINDDQHQYGNWGQAITGDHANLRRVYIGTNGRGIVYGDIGGAPSGSPSPSVSPSASPSLSPSPSPSSSPSPSPSPSSSPSSSPSPSPSPSPSPSRSPSPSASPSPSSSPSPSSSPSSSPSPTPSSSPVSGGVKVQYKNNDSAPGDNQIKPGLQVVNTGSSSVDLSTVTVRYWFTRDGGSSTLVYNCDWAAIGCGNIRASFGSVNPATPT ADTYLQX*

As listed and described in Tables 3 and 2, the isolated AviIIIpolypeptide includes an N-terminal hydrophobic region that functions asa signal peptide, having an amino acid sequence that begins with Met11and extends to about A46; a catalytic domain having significant sequencesimilarity to a GH74 family domain that begins with about A47 andextends to about G786, a carbohydrate binding domain having sequencesimilarity to such type III domains that begins with about V869 andextends to about at least Q956. Variants and derivatives of AviIIIinclude, for example, AviIII polypeptides modified by covalent oraggregative conjugation with other chemical moieties, such as glycosylgroups, polyethylene glycol (PEG) groups, lipids, phosphate, acetylgroups, and the like.

The amino acid sequence of AviIII polypeptides of the invention ispreferably at least about 60% identical, more preferably at least about70% identical, or in some embodiments at least about 90% identical, tothe AviIII amino acid sequence shown above in Table 3 and SEQ ID NO:1.The percentage identity, also termed homology (see definition above) canbe readily determined, for example, by comparing the two polypeptidesequences using any of the computer programs commonly employed for thispurpose, such as the Gap program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,Madison Wis.), which uses the algorithm of Smith and Waterman, 1981,Adv. Appl. Math. 2: 482-489.

Variants and derivatives of the AviIII polypeptide may further include,for example, fusion proteins formed of a AviIII polypeptide and aheterologous polypeptide. Preferred heterologous polypeptides includethose that facilitate purification, oligomerization, stability, orsecretion of the AviIII polypeptides.

AviIII polypeptide variants and derivatives, as used in the descriptionof the invention, can contain conservatively substituted amino acids,meaning that one or more amino acid can be replaced by an amino acidthat does not alter the secondary and/or tertiary structure of thepolypeptide. Such substitutions can include the replacement of an aminoacid, by a residue having similar physicochemical properties, such assubstituting one aliphatic residue (Ile, Val, Leu, or Ala) for another,or substitutions between basic residues Lys and Arg, acidic residues Gluand Asp, amide residues Gln and Asn, hydroxyl residues Ser and Tyr, oraromatic residues Phe and Tyr. Phenotypically silent amino acidexchanges are described more fully in Bowie et al., 1990, Science247:1306-1310. In addition, functional AviIII polypeptide variantsinclude those having amino acid substitutions, deletions, or additionsto the amino acid sequence outside functional regions of the protein,for example, outside the catalytic and carbohydrate binding domains.These would include, for example, the various linker sequences thatconnect functional domains as defined herein.

The AviIII polypeptides of the present invention are preferably providedin an isolated form, and preferably are substantially purified. Thepolypeptides may be recovered and purified from recombinant cellcultures by known methods, including, for example, ammonium sulfate orethanol precipitation, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography, and lectinchromatography. Preferably, high performance liquid chromatography(HPLC) is employed for purification.

Another preferred form of AviIII polypeptides is that of recombinantpolypeptides as expressed by suitable hosts. Furthermore, the hosts cansimultaneously produce other cellulases such that a mixture is producedcomprising a AviIII polypeptide and one or more other cellulases. Such amixture can be effective in crude fermentation processing or otherindustrial processing.

AviIII polypeptides can be fused to heterologous polypeptides tofacilitate purification. Many available heterologous peptides (peptidetags) allow selective binding of the fusion protein to a bindingpartner. Non-limiting examples of peptide tags include 6-His,thioredoxin, hemaglutinin, GST, and the OmpA signal sequence tag. Abinding partner that recognizes and binds to, the heterologous peptidecan be any molecule or compound, including metal ions (for example,metal affinity columns), antibodies, antibody fragments, or any proteinor peptide that preferentially binds the heterologous peptide to permitpurification of the fusion protein.

AviIII polypeptides can be modified to facilitate formation of AviIIIoligomers. For example, AviIII polypeptides can be fused to peptidemoieties that promote oligomerization, such as leucine zippers andcertain antibody fragment polypeptides, for example, Fc polypeptides.Techniques for preparing these fusion proteins are known, and aredescribed, for example, in WO 99/31241 and in Cosman et. al., 2001Immunity 14:123-133. Fusion to an Fc polypeptide offers the additionaladvantage of facilitating purification by affinity chromatography overProtein A or Protein G columns. Fusion to a leucine-zipper (LZ), forexample, a repetitive-heptad repeat, often with four or five leucineresidues interspersed with other amino acids, is described inLandschultz et al., 1988, Science, 240:1759.

It is also envisioned that an expanded set of variants and derivativesof AviIII polynucleotides and/or polypeptides can be generated to selectfor useful molecules, where such expansion is achieved not only byconventional methods such as site-directed mutagenesis (SDM) but also bymore modern techniques, either independently or in combination.

Site-directed-mutagenesis is considered an informational approach toprotein engineering and can rely on high-resolution crystallographicstructures of target proteins and some stratagem for specific amino acidchanges (Van Den Burg, B.; Vriend, G.; Veltman, O. R.; Venema, G.;Eijsink, V. G. H. Proc. Nat. Acad. Sci. U.S. 1998, 95, 2056-2060). Forexample, modification of the amino acid sequence of AviIII polypeptidescan be accomplished as is known in the art, such as by introducingmutations at particular locations by oligonucleotide-directedmutagenesis (Walder et al., 1986, Gene, 42:133; Bauer et al., 1985, Gene37:73; Craik, 1985, BioTechniques, 12-19; Smith et al., 1981, GeneticEngineering: Principles and Methods, Plenum Press; and U.S. Pat. No.4,518,584 and U.S. Pat. No. 4,737,462). SDM technology can also employthe recent advent of computational methods for identifying site-specificchanges for a variety of protein engineering objectives (Helling a, H.W. Nature Structural. Biol. 1998, 5, 525-527).

The more modern techniques include, but are not limited to,non-informational mutagenesis techniques (referred to generically as“directed evolution”). Directed evolution, in conjunction withhigh-throughput screening, allows testing of statistically meaningfulvariations in protein conformation (Arnold, F. H. Nature Biotechnol.1998, 16, 617-618). Directed evolution technology can includediversification methods similar to that described by Crameri A. et al.(1998, Nature 391: 288-291), site-saturation mutagenesis, staggeredextension process (StEP) (Zhao, H.; Giver, is L.; Shao, Z.; Affholter,J. A.; Arnold, F. H. Nature Biotechnol. 1998, 16, 258-262), and DNAsynthesis/reassembly (U.S. Pat. No. 5,965,408).

Fragments of the AviIII polypeptide can be used, for example, togenerate specific anti-AviIII antibodies. Using known selectiontechniques, specific epitopes can be selected and used to generatemonoclonal or polyclonal antibodies. Such antibodies have utility in theassay of AviIII activity as well as in purifying recombinant AviIIIpolypeptides from genetically engineered host cells.

AviIII Polynucleotides

The invention also provides polynucleotide molecules encoding the AviIIIpolypeptides discussed above. AviIII polynucleotide molecules of theinvention include polynucleotide molecules having the nucleic acidsequence shown in Table 4 and SEQ ID NO: 2, polynucleotide moleculesthat hybridize to the nucleic acid sequence of Table 4 and SEQ ID NO: 2under high stringency hybridization conditions (for example, 42° C., 2.5hr., 6×SCC, 0.1% SDS); and polynucleotide molecules having substantialnucleic acid sequence identity with the nucleic acid sequence of Table 4and SEQ ID NO: 2, particularly with those nucleic acids encoding thecatalytic domain, GH74 (from about amino acid A47 to about G786, thecarbohydrate binding domain III (from about amino acid V869 to about atleast Q956.

TABLE 4 AviIII nucleotide sequence. (SEQ ID NO:2)ATGGATCGTTCGGAGAACATCCGTCTGACTATGAGATCACGACGATTGGTATCACTGCTCGCCGCCACTGCGTCGTTCGCCGTGGCCGCCGCTCTGGGAGTTCTGCCCATCGCGATAACGGCTTCTCCTGCGCACGCGGCGACGACTCAGCCGTACACCTGGAGCAACGTGGCGATCGGGGGCGGCGGCTTTGTCGACGGGATCGTCTTCAATGAAGGTGCACCGGGAATTCTGTACGTGCGGACGGACATCGGGGGGATGTATCGATGGGATGCCGCCAACGGGCGGTGGATCCCTCTTCTGGATTGGGTGGGATGGAACAATTGGGGGTACAACGGCGTCGTCAGCATTGCGGCAGACCCGATCAATACTAACAAGGTATGGGCCGCCGTCGGAATGTACACCAACAGCTGGGACCCAAACGACGGAGCGATTCTCCGCTCGTCTGATCAGGGCGCAACGTGGCAAATAACGCCCCTGCCGTTCAAGCTTGGCGGCAACATGCCCGGGCGTGGAATGGGCGAGCGGCTTGCGGTGGATCCAAACAATGACAACATTCTGTATTTCGGCGCCCCGAGCGGCAAAGGGCTCTGGAGAAGCACAGATTCCGGCGCGACCTGGTCCCAGATGACGAACTTTCCGGACGTAGGCACGTACATTGCAAATCCCACTGACACGACCGGCTATCAGAGCGATATTCAAGGCGTCGTCTGGGTCGCTTTCGACAAGTCTTCGTCATCGCTCGGGCAAGCGAGTAAGACCATTTTTGTGGGCGTGGCGGATCCCAATAATCCGGTCTTCTGGAGCAGAGACGGCGGCGCGACGTGGCAGGCGGTGCCGGGTGCGCCGACCGGCTTCATCCCGCACAAGGGCGTCTTTGACCCGGTCAACCACGTGCTCTATATTGCCACCAGCAATACGGGTGGTCCGTATGACGGGAGCTCCGGCGACGTCTGGAAATTCTCGGTGACCTCCGGGACATGGACGCGAATCAGCCCGGTACCTTCGACGGACACGGCCAACGACTACTTTGGTTACAGCGGCCTCACTATCGACCGCCAGCACCCGAACACGATAATGGTGGCAACCCAGATATCGTGGTGGCCGGACACCATAATCTTTCGGAGCACCGACGGCGGTGCGACGTGGACGCGGATCTGGGATTGGACGAGTTATCCCAATCGAAGCTTGCGATATGTGCTTGACATTTCGGCGGAGCCTTGGCTGACCTTCGGCGTACAGCCGAATCCTCCCGTACCCAGTCCGAAGCTCGGCTGGATGGATGAAGCGATGGCAATCGATCCGTTCAACTCTGATCGGATGCTCTACGGAACAGGCGCGACGTTGTACGCAACAAATGATCTCACGAAGTGGGACTCCGGCGGCCAGATTCATATCGCGCCGATGGTCAAAGGATTGGAGGAGACGGCGGTAAACGATCTCATCAGCCCGCCGTCTGGCGCCCCGCTCATCAGCGCTCTCGGAGACCTCGGCGGCTTCACCCACGCCGACGTTACTGCCGTGCCATCGACGATCTTCACGTCACCGGTGTTCACGACCGGCACCAGCGTCGACTATGCGGAATTGAATCCGTCGATCATCGTTCGCGCTGGAAGTTTCGATCCATCGAGCCAACCGAACGACAGGCACGTCGCGTTCTCGACAGACGGCGGCAAGAACTGGTTCCAAGGCAGCGAACCTGGCGGGGTGACGACGGGCGGCACCGTCGCCGCATCGGCCGACGGCTCTCGTTTCGTCTGGGCTCCCGGCGATCCCGGTCAGCCTGTGGTGTACGCAGTCGGATTTGGCAACTCCTGGGCTGCTTCGCAAGGTGTTCCCGCCAATGCCCAGATCCGCTCAGACCGGGTGAATCCAAAGACTTTCTATGCCCTATCCAATGGAACCTTCTATCGAAGCACGGACGGCGGCGTGACATTCCAACCGGTCGCGGCCGGTCTTCCGAGCAGCGGTGCCGTCGGTGTCATGTTCCACGCGGTGCCTGGAAAAGAAGGCGATCTGTGGCTCGCTGCATCGAGCGGGCTTTACCACTCAACCAATGGCGGCAGCAGTTGGTCTGCAATCACCGGCGTATCCTCCGCGGTGAACGTGGGATTTGGTAAGTCTGCGCCCGGGTCGTCATACCCAGCCGTCTTTGTCGTCGGCACGATCGGAGGCGTTACGGGGGCGTACCGCTCCGACGACTGTGGGACGACCTGGGTACTGATCAATGATGACCAGCACCAATACGGAAATTGGGGACAAGCAATCACCGGTGACCACGCGAATTTACGGCGGGTGTACATAGGCACGAACGGCCGTGGAATTGTATACGGGGACATTGGTGGTGCGCCGTCCGGATCGCCGTCTCCGTCGGTGAGTCCGTCGGCTTCGCCGAGCCTGAGCCCGAGCCCGAGCCCGAGCAGCTCGCCATCGCCGTCGCCGTCGCCGAGCTCGAGTCCATCCTCGTCGCCGTCTCCGTCGCCGTCACCATCGCCGAGTCCGTCTCGGTCTCCGTCACCATCGGCGTCGCCGAGCCCGTCTTCGTCACCGAGCCCGTCTTCGTCACCGTCTTCGTCGCCGAGCCCAACGCCGTCGTCGTCGCCGGTGTCGGGTGGGGTGAAGGTGCAGTATAAGAATAATGATTCGGCGCCGGGTGATAATCAGATCAAGCCGGGTTTGCAGGTGGTGAATACCGGGTCGTCGTCGGTGGATTTGTCGACGGTGACGGTGCGGTACTGGTTCACCCGGGATGGTGGCTCGTCGACACTGGTGTACAACTGTGACTGGGCGGCGATCGGGTGTGGGAATATCCGCGCCTCGTTCGGCTCGGTGAACCCGGCGACGCCGACGGCGGACACCTACCTGCAGN*

The AviIII polynucleotide molecules of the invention are preferablyisolated molecules encoding the AviIII polypeptide having an amino acidsequence as shown in Table 3 and SEQ ID NO:1, as well as derivatives,variants, and useful fragments of the AviIII polynucleotide. The AviIIIpolynucleotide sequence can include deletions, substitutions, oradditions to the nucleic acid sequence of Table 4 and SEQ ID NO: 2.

The AviIII polynucleotide molecule of the invention can be cDNA,chemically synthesized DNA, DNA amplified by PCR, RNA, or combinationsthereof. Due to the degeneracy of the genetic code, two DNA sequencesmay differ and yet encode identical amino acid sequences. The presentinvention thus provides an isolated polynucleotide molecule having aAviIII nucleic acid sequence encoding AviIII polypeptide, where thenucleic acid sequence encodes a polypeptide having the complete aminoacid sequences as shown in Table 3 and SEQ ID NO: 1, or variants,derivatives, and fragments thereof.

The AviIII polynucleotides of the invention have a nucleic acid sequencethat is at least about 60% identical to the nucleic acid sequence shownin Table 4 and SEQ ID NO: 2, in some embodiments at least about 70%identical to the nucleic acid sequence shown in Table 4 and SEQ ID NO:2, and in other embodiments at least about 90% identical to the nucleicacid sequence shown in Table 4 and SEQ ID NO: 2. Nucleic acid sequenceidentity is determined by known methods, for example by aligning twosequences in a software program such as the BLAST program (See Altschul,S. F. et al. (1990) J. Mol. Biol. 215:403-410).

The AviIII polynucleotide molecules of the invention also includeisolated polynucleotide molecules' having a nucleic acid sequence thathybridizes under high stringency conditions (as defined above) to a thenucleic acid sequence shown in Table 4 and SEQ ID NO: 2. Hybridizationof the polynucleotide is to about 15 contiguous nucleotides, or about 20contiguous nucleotides, and in other embodiments about 30 contiguousnucleotides, and in still other embodiments about 100 contiguousnucleotides of the nucleic acid sequence shown in Table 4 and SEQ ID NO:2.

Useful fragments of the AviIII-encoding polynucleotide moleculesdescribed herein, include probes and primers. Such probes and primerscan be used, for example, in PCR methods to amplify and detect thepresence of AviIII polynucleotides in vitro, as well as in Southern andNorthern blots for analysis of AviIII. Cells expressing the AviIIIpolynucleotide molecules of the invention can also be identified by theuse of such probes. Methods for the production and use of such primersand probes are known. For PCR, 5′ and 3′ primers corresponding to aregion at the termini of the AviIII polynucleotide molecule can beemployed to isolate and amplify the AviIII polynucleotide usingconventional techniques.

Other useful fragments of the AviIII polynucleotides include antisenseor sense oligonucleotides comprising a single-stranded nucleic acidsequence capable of binding to a target AviIII mRNA (using a sensestrand), or DNA (using an antisense strand) sequence.

Vectors and Host Cells:

The present invention also provides vectors containing thepolynucleotide molecules of the invention, as well as host cellstransformed with such vectors. Any of the polynucleotide molecules ofthe invention may be contained in a vector, which generally includes aselectable marker and an origin of replication, for propagation in ahost. The vectors further include suitable transcriptional ortranslational regulatory sequences, such as those derived from amammalian, microbial, viral, or insect genes, operably linked to theAviIII polynucleotide molecule. Examples of such regulatory sequencesinclude transcriptional promoters, operators, or enhancers, mRNAribosomal binding sites, and appropriate sequences which controltranscription and translation. Nucleotide sequences are operably linkedwhen the regulatory sequence functionally relates to the DNA encodingthe target protein. Thus, a promoter nucleotide sequence is operablylinked to a AviIII DNA sequence if the promoter nucleotide sequencedirects the transcription of the AviIII sequence.

Selection of suitable vectors for the cloning of AviIII polynucleotidemolecules encoding the target AviIII polypeptides of this invention willdepend upon the host cell in which the vector will be transformed, and,where applicable, the host cell from which the target polypeptide is tobe expressed. Suitable host cells for expression of AviIII polypeptidesinclude prokaryotes, yeast, and higher eukaryotic cells, each of whichis discussed below.

The AviIII polypeptides to be expressed in such host cells may also befusion proteins that include regions from heterologous proteins. Asdiscussed above, such regions may be included to allow, for example,secretion, improved stability, or facilitated purification of the AviIIIpolypeptide. For example, a nucleic acid sequence encoding anappropriate signal peptide can be incorporated into an expressionvector. A nucleic acid sequence encoding a signal peptide (secretoryleader) may be fused in-frame to the AviIII sequence so that AviIII istranslated as a fusion protein comprising the signal peptide. A signalpeptide that is functional in the intended host cell promotesextracellular secretion of the AviIII polypeptide. Preferably, thesignal sequence will be cleaved from the AviIII polypeptide uponsecretion of AviIII from the cell. Non-limiting examples of signalsequences that can be used in practicing the invention include the yeastI-factor and the honeybee melatin leader in Sf9 insect cells.

Suitable host cells for expression of target polypeptides of theinvention include prokaryotes, yeast, and higher eukaryotic cells.Suitable prokaryotic hosts to be used for the expression of thesepolypeptides include bacteria of the genera Escherichia, Bacillus, andSalmonella, as well as members of the genera Pseudomonas, Streptomyces,and Staphylococcus. For expression in prokaryotic cells, for example, inE. coli, the polynucleotide molecule encoding AviIII polypeptidepreferably includes an N-terminal methionine residue to facilitateexpression of the recombinant polypeptide. The N-terminal Met mayoptionally be cleaved from the expressed polypeptide.

Expression vectors for use in prokaryotic hosts generally comprise oneor more phenotypic selectable marker genes. Such genes encode, forexample, a protein that confers antibiotic resistance or that suppliesan auxotrophic requirement. A wide variety of such vectors are readilyavailable from commercial sources. Examples include pSPORT vectors, pGEMvectors (Promega, Madison, Wis.), pPROEX vectors (LTI, Bethesda, Md.),Bluescript vectors (Stratagene), and pQE vectors (Qiagen).

AviIII can also be expressed in yeast host cells from genera includingSaccharomyces, Pichia, and Kluveromyces. Preferred yeast hosts are S.cerevisiae and P. pastoris. Yeast vectors will often contain an originof replication sequence from a 2 T yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene. Vectors replicable in both yeast and E. coli(termed shuttle vectors) may also be used. In addition to theabove-mentioned features of yeast vectors, a shuttle vector will alsoinclude sequences for replication and selection in E. coli. Directsecretion of the target polypeptides expressed in yeast hosts may beaccomplished by the inclusion of nucleotide sequence encoding the yeastI-factor leader sequence at the 5′ end of the AviIII-encoding nucleotidesequence.

Insect host cell culture systems can also be used for the expression ofAviIII polypeptides. The target polypeptides of the invention arepreferably expressed using a baculovirus expression system, asdescribed, for example, in the review by Luckow and Summers, 1988Bio/Technology 6:47.

The choice of a suitable expression vector for expression of AviIIIpolypeptides of the invention will depend upon the host cell to be used.Examples of suitable expression vectors for E. coli include pET, pUC,and similar vectors as is known in the art. Preferred vectors forexpression of the AviIII polypeptides include the shuttle plasmid pIJ702for Streptomyces lividans, pGAPZalpha-A, B, C and pPICZalpha-A, B, C(Invitrogen) for Pichia pastoris, and pFE-1 and pFE-2 for filamentousfungi and similar vectors as is known in the art.

Modification of a AviIII polynucleotide molecule to facilitate insertioninto a particular vector (for example, by modifying restriction sites),ease of use in a particular expression system or host (for example,using preferred host codons), and the like, are known and arecontemplated for use in the invention. Genetic engineering methods forthe production of AviIII polypeptides include the expression of thepolynucleotide molecules in cell free expression systems, in cellularhosts, in tissues, and in animal models, according to known methods.

Compositions

The invention provides compositions containing a substantially purifiedAviIII polypeptide of the invention and an acceptable carrier. Suchcompositions are administered to biomass, for example, to degrade thecellulose in the biomass into simpler carbohydrate units and ultimately,to sugars. These released sugars from the cellulose are converted intoethanol by any number of different catalysts. Such compositions may alsobe included in detergents for removal, for example, of cellulosecontaining stains within fabrics, or compositions used in the pulp andpaper industry, to address conditions associated with cellulose content.Compositions of the present invention can be used in stonewashing jeanssuch as is well known in the art. Compositions can be used in thebiopolishing of cellulosic fabrics, such as cotton, linen, rayon andLyocell.

The invention provides pharmaceutical compositions containing asubstantially purified AviIII polypeptide of the invention and ifnecessary a pharmaceutically acceptable carrier. Such pharmaceuticalcompositions are administered to cells, tissues, or patients, forexample, to aid in delivery or targeting of other pharmaceuticalcompositions. For example, AviIII polypeptides may be used wherecarbohydrate-mediated liposomal interactions are involved with targetcells. Sihorkar, V. et al. (2001), J. Pharmacy & Pharmaceutical SciencesMay-August 4(2): 138-58.

The invention also provides reagents, compositions, and methods that areuseful for analysis of AviIII activity and for the analysis of cellulosebreakdown.

Compositions of the present invention may also include other knowncellulases, and preferably, other known thermal tolerant cellulases forenhanced treatment of cellulose.

Antibodies

The polypeptides of the present invention, in whole or in part, may beused to raise polyclonal and monoclonal antibodies that are useful inpurifying AviIII, or detecting AviIII polypeptide expression, as well asa reagent tool for characterizing the molecular actions of the AviIIIpolypeptide. Preferably, a peptide containing a unique epitope of theAviIII polypeptide is used in preparation of antibodies, usingconventional techniques. Methods for the selection of peptide epitopesand production of antibodies are known. See, for example, Antibodies: ALaboratory Manual, Harlow and Land (eds.), 1988 Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al.(eds.), 1980 Plenum Press, New York.

Assays

Agents that modify, for example, increase or decrease, AviIII hydrolysisor degradation of cellulose can be identified, for example, by assay ofAviIII cellulase activity and/or analysis of AviIII binding to acellulose substrate. Incubation of cellulose in the presence of AviIIIand in the presence or absence of a test agent and correlation ofcellulase activity or carbohydrate binding permits screening of suchagents. For example, cellulase activity and binding assays may beperformed in a manner similar to those described in Irwin et al., J.Bacteriology 180(7): 1709-1714 (April 1998).

The AviIII stimulated activity is determined in the presence and absenceof a test agent and then compared. A lower AviIII activated testactivity in the presence of the test agent, than in the absence of thetest agent, indicates that the test agent has decreased the activity ofthe AviIII. A higher AviIII activated test activity in the presence ofthe test agent than in the absence of the test agent indicates that thetest agent has increased the activity of the AviIII. Stimulators andinhibitors of AviIII may be used to augment, inhibit, or modify AviIIImediated activity, and therefore may have potential industrial uses aswell as potential use in the further elucidation of AviIII's molecularactions.

Therapeutic Applications

The AviIII polypeptides of the invention are effective in adding indelivery or targeting of other pharmaceutical compositions within ahost. For example, AviIII polypeptides may be used wherecarbohydrate-mediated liposomal interactions are involved with targetcells. Sihorkar, V. et al. (2001), J. Pharm Pharm Sci. May-August 4(2):138-58.

AviIII polynucleotides and polypeptides, including vectors expressingAviIII, of the invention can be formulated as pharmaceuticalcompositions and administered to a host, preferably mammalian host,including a human patient, in a variety of forms adapted to the chosenroute of administration. The compounds are preferably administered incombination with a pharmaceutically acceptable carrier, and may becombined with or conjugated to specific delivery agents, includingtargeting antibodies and/or cytokines.

AviIII can be administered by known techniques, such as orally,parentally (including subcutaneous injection, intravenous,intramuscular, intrasternal or infusion techniques), by inhalationspray, topically, by absorption through a mucous membrane, or rectally,in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants or vehicles.Pharmaceutical compositions of the invention can be in the form ofsuspensions or tablets suitable for oral administration, nasal sprays,creams, sterile injectable preparations, such as sterile injectableaqueous or oleagenous suspensions or suppositories.

For oral administration as a suspension, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can contain microcrystalline cellulose forimparting bulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweeteners or flavoringagents. As immediate release tablets, the compositions can containmicrocrystalline cellulose, starch, magnesium stearate and lactose orother excipients, binders, extenders, disintegrants, diluents andlubricants known in the art.

For administration by inhalation or aerosol, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can be prepared as solutions in saline,using benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons or othersolubilizing or dispersing agents known in the art.

For administration as injectable solutions or suspensions, thecompositions can be formulated according to techniques well-known in theart, using suitable dispersing or wetting and suspending agents, such assterile oils, including synthetic mono- or diglycerides, and fattyacids, including oleic acid.

For rectal administration as suppositories, the compositions can beprepared by mixing with a suitable non-irritating excipient, such ascocoa butter, synthetic glyceride esters or polyethylene glycols, whichare solid at ambient temperatures, but liquefy or dissolve in the rectalcavity to release the drug.

Preferred administration routes include orally, parenterally, as well asintravenous, intramuscular or subcutaneous routes. More preferably, thecompounds of the present invention are administered parenterally, i.e.,intravenously or intraperitoneally, by infusion or injection.

Solutions or suspensions of the compounds can be prepared in water,isotonic saline (PBS) and optionally mixed with a nontoxic surfactant.Dispersions may also be prepared in glycerol, liquid polyethylene,glycols, DNA, vegetable oils, triacetin and mixtures thereof. Underordinary conditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

The pharmaceutical dosage form suitable for injection or infusion usecan include sterile, aqueous solutions or dispersions or sterile powderscomprising an active ingredient which are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions.In all cases, the ultimate dosage form should be sterile, fluid andstable under the conditions of manufacture and storage. The liquidcarrier or vehicle can be a solvent or liquid dispersion mediumcomprising, for example, water, ethanol, a polyol such as glycerol,propylene glycol, or liquid polyethylene glycols and the like, vegetableoils, nontoxic glyceryl esters, and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the formation ofliposomes, by the maintenance of the required particle size, in the caseof dispersion, or by the use of nontoxic surfactants. The prevention ofthe action of microorganisms can be accomplished by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, buffers, or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the inclusion in thecomposition of agents delaying absorption—for example, aluminummonosterate hydrogels and gelatin.

Sterile injectable solutions are prepared by incorporating the compoundsin the required amount in the appropriate solvent with various otheringredients as enumerated above and, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying techniques, which yield a powder of theactive ingredient plus any additional desired ingredient present in thepreviously sterile-filtered solutions.

INDUSTRIAL APPLICATIONS

The AviIII polypeptides of the invention are effective cellulases. Inthe methods of the invention, the cellulose degrading effects of AviIIIare achieved by treating biomass at a ratio of about 1 to about 50, orabout 1:40, 1:35, 1:30, 1:25, 1:20 or even about 1:70 in somepreparations of the AViIII of AviII:biomass. AviIII may be used underextreme conditions, for example, elevated temperatures and acidic pH.Treated biomass is degraded into simpler forms of carbohydrates, and insome cases glucose, which is then used in the formation of ethanol orother industrial chemicals, as is known in the art. Other methods areenvisioned to be within the scope of the present invention, includingmethods for treating fabrics to remove cellulose-containing stains andother methods already discussed. AviIII polypeptides can be used in anyknown application currently utilizing a cellulase, all of which arewithin the scope of the present invention.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Molecular Cloning of AviIII

Genomic DNA was isolated from Acidothermus cellulolyticus and purifiedby banding on cesium chloride gradients. Genomic DNA was partiallydigested with Sau 3A and separated on agarose gels. DNA fragments in therange of 9-20 kilobase pairs were isolated from the gels. This purifiedSau 3A digested genomic DNA was ligated into the Bam H1 acceptor site ofpurified EMBL3 lambda phage arms (Clontech, San Diego, Calif.). PhageDNA was packaged according to the manufacturer's specification andplated with E. Coli LE392 in top agar which contained the solublecellulose analog, carboxymethylcellulose (CMC). The plates wereincubated overnight (12-24 hours) to allow transfection, bacterialgrowth, and plaque formation. Plates were stained with Congo Redfollowed by destaining with 1 M NaCl. Lambda plaques harboringendoglucanase clones showed up as unstained plaques on a red background.

Lambda clones which screened positive on CMC-Congo Red plates werepurified by successive rounds of picking, plating and screening.Individual phage isolates were named SL-1, SL-2, SL-3, and SL-4.Subsequent subcloning efforts employed the SL-3 clone which contained anapproximately 14.2 kilobase fragment of Acidothermus cellulolyticusgenomic DNA.

Template DNA was constructed using a 9 kilobase Bam H1 fragment obtainedfrom the 14.2 kilobase lambda clone SL-3 prepared from Acidothermuscellulolyticus genomic DNA. The 9 kilobase Bam H1 fragment from SL-3 wassubcloned into pDR540 to generate a plasmid NREL501. NREL501 wassequenced by the primer walking method as is known in the art. NREL501was then subcloned into pUC19 using restriction enzymes Pst I and Eco RIand transformed into E. coli XL1-blue (Stratagene) for the production oftemplate DNA for sequencing. Each subclone was sequenced from both theforward and reverse directions. DNA for sequencing was prepared from anovernight growth in 500 mL LB broth using a megaprep DNA purificationkit from Promega. The templated DNA was PEG precipitated and suspendedin de-ionized water and adjusted to a final concentration of 0.25milligrams/mL.

Custom primers were designed by reading upstream known sequence andselecting segments of an appropriate length to function, as is wellknown in the art. Primers for cycle sequencing were synthesized at theMacromolecular Resources Facility located at Colorado State Universityin Fort Collins, Colo. Typically the sequencing primers were 26 to 30nucleotides in length, but were sometimes longer or shorter toaccommodate a melting temperature appropriate for cycle sequencing. Thesequencing primers were diluted in de-ionized water, the concentrationmeasured using UV absorbance at 260 nm, and then adjusted to a finalconcentration of 5 pmol/microL.

Templates and sequencing primers were shipped to the Iowa StateUniversity DNA Sequencing Facility at Ames, Iowa for sequencing usingstandard chemistries for cycle sequencing. In some cases, regions of thetemplate that sequenced poorly using the standard protocols and dyeterminators were repeated with the addition of 2 microL DMSO and byusing nucleotides optimized for the sequencing of high GC content DNA.An inverse PCR technique known in the art was applied to continuesequencing the genomic DNA, and a primer walking method was used tosequence the large PCR products. Each PCR fragment was sequenced fromboth strands, using high fidelity commercial DNA polymerase.

Sequencing data from primer walking and subclones were assembledtogether to verify that all SL-3 regions had been sequenced from bothstrands. An open reading frame (ORF) was found in the 9 kilobase Bam H1fragment, C-terminal of E1 (U.S. Pat. No. 5,536,655), termed AviIII. AnORF of about 3 kb (SEQ ID NO:2) and deduced amino acid sequence (SEQ IDNO:1) are shown in Tables 3 and 4. The amino acid sequence predicted bySEQ ID NO:1 was determined to have significant homology to knowncellulases, as is shown below in Example 2 and Table 5.

The amino acid sequence represents a novel member of the family ofproteins with cellulase activity. Due to the source of isolation, fromthe thermophilic Acidothermus cellulolyticus, AviIII is a novel memberof cellulases with properties including thermal tolerance. It is alsoknown that thermal tolerant enzymes may have other properties (seedefinition above).

Example 2 AviIII Includes a GH74 Catalytic Domain

Sequence alignments and comparisons of the amino acid sequences of theAcidothermus cellulolyticus AviIII catalytic domain (approximately aminoacids 47 to 786) and Aspergillus aculeatus Avicelase III (endoglucanase)polypeptides were prepared, using the ClustalW program. (See Thompson J.D. et al. (1994), Nucleic Acids Res. 22:4673-4680). An examination ofthe amino acid sequence alignment of the GH74 domain indicates that theamino acid sequence of AviIII catalytic domain is homologous to theamino acid sequence of a known GH74 family catalytic domain forAspergillus aculeatus Avicelase III (endoglucanase) (see Table 5). InTable 5, the notations are as follows: an asterisk “*” indicatesidentical or conserved residues in all sequences in the alignment; acolon “:” indicates conserved substitutions; a period “.” indicatessemi-conserved substitutions; and a hyphen “-” indicates a gap in thesequence. The amino acid sequence predicted for the AviIII GH74 domainis approximately 46% identical to the Aspergillus aculeatus Avicelase(III) (endoglucanase) GH74 domain, indicating that the AviIII catalyticdomain is a member of the GH74 family (Henrissat et al., (1991) supra).

TABLE 5 Multiple amino acid sequence alignment of a AviIII catalyticdomain and polypeptides with Glycoside Hydrolase Family 74 catalyticdomains. Multialignment of related Glycoside Hydrolase Family 74catalytic domain GH74_Ace: Acidothermus cellulolyticus AviIII catalyticdomain GH74 AviIII_Aac: Aspergillus aculeatus Avicelase III(endoglucanase). Gene- Bank Acc. #BAA29031 GH74_AceATTQPYTWSNVAIGGGG-FVDGIVFNEGAPGILYVRTDIGGMYRWDAANGRWIPLLDWVG AviIII_AacAASQAYTWKNVVTGGGGGFTPGIVFNPSAKGVAYARTDIGGAYRLNSDD-TWTPLMDWVG*::*.***.**. **** *. ***** .* *: *.****** ** :: :  * **:**** GH74_AceWNNWGYNGVVSIAADPINTNKVWAAVGMYTNSWDPNDGAILRSSDQGATWQITPLPFKLG AviIII_AacNDTWHDWGIDALATDPVDTDRVYVAVGMYTNEWDPNVGSILRSTDQGDTWTETKLPFKVG :.*   *: ::*:**::*::*:.*******.**** *:****:*** **  * ****:* GH74_AceGNMPGRGMGERLAVDPNNDNILYFGAPSGKGLWRSTDSGATWSQMTNFPDVGTYIANPTD AviIII_AacGNMPGRGMGERLAVDPNKNSILYFGARSGHGLWKSTDYGATWSNVTSFTWTGTYFQDSSS*****************::.****** **:***:*** *****::*.*. .***: :.:. GH74_AceTTGYQSDIQGVVWVAFDKSSSSLGQASKTIFVGVADPNNPVFWSRDGGATWQAVPGAP-T AviIII_AacT--YTSDPVGIAWVTFDSTSGSSGSATPRIFVGVADAGKSVFKSEDAGATWAWVSGEPQY*  * **  *:.**:**.:*.* *.*:  *******..:.** *.*.****  *.* * GH74_AceGFIPHKGVFDPVNHVLYIATSNTGGPYDGSSGDVWKFSVTSGTWTRISPVPSTDTANDYF AviIII_AacGFLPHKGVLSPEEKTLYISYANGAGPYDGTNGTVHKYNITSGVWTDISP---TSLASTYY**:*****:.* ::.***: :* .*****:.* * *:.:***.** ***   *. *. *: GH74_AceGYSGLTIDRQHPNTIMVATQISWWPDTIIFRSTDGGATWTRIWDWTSYPNRSLRYVLDIS AviIII_AacGYGGLSVDLQVPGTLMVAALNCWWPDELIFRSTDSGATWSPIWEWNGYPSINYYYSYDIS**.**::* * *.*:***:  .**** :******.****: **:*..**. .  *  *** GH74_AceAEPWLTFGVQPNPPVPSPKLGWMDEAMAIDPFNSDRMLYGTGATLYATNDLTKWDSGGQI AviIII_AacNAPWIQDTTSTDQFP--VRVGWMVEALAIDPFDSNHWLYGTGLTVYGGHDLTNWDSKHNV  **:   ...:      ::*** **:*****:*:: ***** *:*. :***:***  :: GH74_AceHIAPMVKGLEETAVNDLISPPSGAPLISALGDLGGFTHADVTAVPSTIFTSPVFTTGTSV AviIII_AacTVKSLAVGIEEMAVLGLITPPGGPALLSAVGDDGGFYHSDLDAAPNQAYHTPTYGTTNGI : .:. *:** ** .**:**.*..*:**:** *** *:*: *.*.  : :*.: * ..: GH174_AceDYAELNPSIIVRAGSFDPSSQPNDRHVAFSTDGGKNWFQGSEPGGVTTGGTVAASADGSR AviIII_AacDYAGNKPSNIVRSGASDDYP-----TLALSSNFGSTWYADYAASTSTGTGAVALSADGDT***  :** ***:*: *  .      :*:*:: *..*: .  ..  *  *:** ****. GH74_AceFVWAPGDPGQPVVYAVGFGNSWAASQGVPANAQIRSDRVNPKTFYALSNGTFYRSTDGGV AviIII_AacVLLMSSTSGALVSKSQG---TLTAVSSLPSGAVIASDKSDNTVFYGGSAGAIYVSKNTAT.:  .. .*  *  : *   : :* ..:*:.* * **: : ..**. * *::* *.: .. GH74_AceTFQPVAAGLPSSGAVGVMFHAVPGKEGDLWLAASSGLYHSTNGGSSWSAI-TGVSSAVNV AviIII_AacSFTKTVS-LGSSTTVNAIR-AHPSIAGDVWASTDKGLWHSTDYGSTFTQIGSGVTAGWSF:*  ..: * ** :*..:  * *.  **:* ::..**:***: **::: * :**::. .. GH74_AceGFGKSAPGSSYPAVFVVGTIGGVTGAYRSDDCGTTWVLINDDQHQYGN-WGQAITGDHAN AviIII_AacGFGKASSTGSYVVIYGFFTIDGAAGLFKSEDAGTNWQVISDASHGFGSGSANVVNGDLQT****::. .** .:: . **.*.:* ::*:*.**.* :*.* .* :*.  .:.:.**  . GH74_AceLRRVYIGTNGRGIVYGDIGGAPSG AviIII_Aac YGRVFRGHERPGHLLRQSQREPAG  **: * :  * :  :    *:*

Example 3 Mixed Domain GH74, CBD II, CBD III Genes and HybridPolypeptides

From the putative locations of the domains in the AviIII cellulasesequence given above and in comparable cloned cellulase sequences fromother species, one can separate individual domains and combine them withone or more domains from different sequences. The significant similaritybetween cellulase genes permit one by recombinant techniques to arrangeone or more domains from the Acidothermus cellulolyticus AviIIIcellulase gene with one or more domains from a cellulase gene from oneor more other microorganisms. Other representative endoglucanase genesinclude Bacillus polymyxa beta-(1,4) endoglucanase (Baird et al, Journalof Bacteriology, 172: 1576-86 (1992)) and Xanthomonas campestrisbeta-(1,4)-endoglucanase A (Gough et al, Gene 89:53-59 (1990)). Theresult of the fusion of any two or more domains will, upon expression,be a hybrid polypeptide. Such hybrid polypeptides can have one or morecatalytic or binding domains. For ease of manipulation, recombinanttechniques may be employed such as the addition of restriction enzymesites by site-specific mutagenesis. If one is not using one domain of aparticular gene, any number of any type of change including completedeletion may be made in the unused domain for convenience ofmanipulation.

It is understood for purposes of this disclosure, that various changesand modifications may be made to the invention that are well within thescope of the invention. Numerous other changes may be made which willreadily suggest themselves to those skilled in the art and which areencompassed in the spirit of the invention disclosed herein and asdefined in the appended claims.

This specification contains numerous citations to references such aspatents, patent applications, and publications. Each is herebyincorporated by reference for all purposes.

1. An isolated polynucleotide molecule encoding a polypeptide comprisingthe sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 havingcellulase activity.
 2. The isolated polynucleotide molecule of claim 1,further comprising a nucleic acid sequence encoding a heterologousprotein in frame with the polynucleotide molecule.
 3. The isolatedpolynucleotide molecule of claim 2, wherein the heterologous protein isa peptide tag.
 4. The isolated polynucleotide molecule of claim 3,wherein the peptide tag is 6-His, thioredoxin, hemaglutinin, GST, orOmpA signal sequence tag.
 5. The isolated polynucleotide molecule ofclaim 2, wherein the heterologous protein is capable of binding acarbohydrate molecule.
 6. The isolated polynucleotide molecule of claim1, operably linked to a transcriptional or translational regulatorysequence.
 7. The isolated polynucleotide molecule of claim 6, whereinthe transcriptional or translational regulatory sequence comprises atranscriptional promoter or enhancer.
 8. A vector comprising thepolynucleotide molecule of claim
 7. 9. A host cell geneticallyengineered to express a polypeptide from the polynucleotide molecule ofclaim
 7. 10. The host cell of claim 9, wherein the host cell is a plantcell.
 11. The host cell of claim 9, wherein the host cell is a fungus.12. The host cell of claim 9, wherein the host cell is a bacterial cell.13. The host cell of claim 9, wherein the host cell is a yeast.
 14. Amethod for producing the polypeptide encoded by the polynucleotidemolecule of claim 7, the method comprising: incubating a host cellgenetically engineered to express the polynucleotide molecule of claim 7under conditions that allow the polypeptide to be produced.
 15. Themethod of claim 14, further comprising the step of: isolating thepolypeptide from the incubated host cells.
 16. The method of claim 14,wherein the host cell is a plant cell.
 17. The method of claim 14,wherein the host cell is a bacterial cell.
 18. The method of claim 14,wherein the host cell is genetically engineered to express a selectablemarker.
 19. The polynucleotide molecule of claim 1, wherein thepolypeptide comprises the sequence of SEQ ID NO.
 1. 20. Thepolynucleotide molecule of claim 1, wherein the polypeptide comprises afragment of SEQ ID NO. 1 having cellulase activity.